This invention relates to a method involving blood leucocyte apheresis for treatment of disease.
For some time, therapeutic apheresis has been known as an alternative strategy for the management of disorders. The technique involves the removal of cells or proteins that are implicated in the pathogenesis of the disorder, for example rheumatoid factor, immune complexes and other humoral mediators in the plasma (1, 2). Procedures employing selective removal of blood cells or plasma components, without the use of replacement fluids, are associated with minimal side effects (1). An outpatient approach is possible, which is more convenient for the patient and reduces the cost of the procedure (2). Many of the unacceptable side-effects of chronic drug therapy are also reduced by use of the technique.
In EP-A-0-319961 there is described a method for the selective removal of granulocytes from blood by bringing the blood into contact with an adsorbent carrier having a higher affinity for granulocytes than for lymphocytes. That invention is concerned with cancer treatment. An apparatus for carrying out the method is also described in that application. A similar apparatus is described in U.S. Pat. No. 5,725,768.
In JP 0193 481 and U.S. Pat. No. 5,567,443 there is described a method for the treatment of inflammatory disease which comprises bringing blood from a patient into contact with an adsorbent carrier that has an affinity for inflammatory cells (granulocytes and monocytes) which is greater than its affinity for lymphocytes. The method is reported to be useful for treating acute respiratory distress syndrome, rheumatoid arthritis, autoimmune diseases, allergic diseases and reflow disorders following myocardial infarction.
AIDS is a life-threatening immunodeficiency condition caused by HIV (human immunodeficiency virus). After confirmation and onset of HIV infection, the mortality rate is very high. No effective treatment strategy has been established yet for the treatment of AIDS and, owing to a long latent period, it is also difficult to establish an effective measure for the prevention of infection.
After infection with HIV, anti-HIV antibody appears in the blood, generally within 6 to 8 weeks. Deficiency of or disturbance to the immune system proceeds, leading eventually to AIDS. For convenience, the intermediate stage is classified as AIDS related complex (ARC). Certain cells infected with HIV harbour the virus and act as reservoirs of the HIV. Such cells include mononuclear cells (monocytes/macrophages) and resting CD4+ lymphocytes. While the virus is inside those cells, it is protected from clearance by the immune system. The virus is also shielded from contact with administered drugs so effective treatment of the condition is thus difficult.
The main symptoms of AIDS include fever, persistent systemic lymphadenoma, weight loss (cachexia) and diarrhoea. In addition, opportunistic infectious diseases and malignant tumours may develop and profoundly aggravate the patient""s condition. Neuropathy may also become a serious problem and, depending on the patient, a disease of the cranial nervous system may also appear.
Based on an immunological consideration of full blown HIV-induced disease (AIDS), the condition is characterised by a profound impairment of the immune response, including decline in CD4+ T lymphocytes, dysregulated cytokine production, increased production of proinflammatory or immunosuppressive cytokines such as TNF-xcex1, IL-1xcex2, IL-6 and IL-10 by peripheral blood cells (3-6) including monocytes (3) and also by granulocytes (4) and enhanced susceptibility of immune cells to apoptosis. HIV antigens such as gp120 bound to immune complexes are believed to induce apoptosis of CD4+ cells (7-12). TNF-xcex1, IL-1xcex2, IL-6 are reported to promote HIV replication (3). At the same time, there is a profound impairment of the ability of monocytes from HIV-infected patients to generate IL-12 (an immunoregulator cytokine). Thus, in healthy individuals, monocytes mainly produce IL-12, whereas in HIV-infected individuals, monocytes produce mainly IL-10. IL-10 was shown to have a pivotal role in the aforementioned changes in the production of cytokines (5, 6). In this context, it is also important to indicate that IL-2 and IL-12 reduce, whereas IL-10 increases susceptibility of CD4+ cells of HIV-infected patients to apoptosis (13-17).
Currently, the treatment strategy for HIV infection consists primarily of highly active antiretroviral drug therapy (HAART) which includes a protease inhibitor. However, whilst in some patients, HAART has been shown to increase CD4+ cell count and to suppress plasma HIV viral load to undetectable levels, as yet no drug treatment is known to affect cellular reservoirs of HIV, for example, in cells including mononuclear cells (monocytes/macrophages) and resting CD4+ lymphocytes. These cells serve as sites for an HIV reservoir and as sites for HIV replication and dissemination. This means that when drug treatment is ceased, there is a very strong possibility of relapse attributable to HIV propagation from the aforementioned reservoir cells and de navo infection. In the light of this understanding and for complete eradication of HIV, it is considered to be essential to eliminate the infected resting cells (latent HIV reservoirs). However, at present, there is no drug that can affect the cellular HIV reservoirs, the HIV-1 proviral DNA load in the latently infected mononuclear cells (monocytes/macrophages) and resting CD4+ lymphocytes.
The hepatitis C virus (HCV) infects cells, for example liver cells, and subsequently harbours in those cells and in leucocytes during an apparently latent period. At present, there is no drug that can affect the cellular HCV reservoirs in those latently infected cells.
Mycobacterium leprae infects and then remains in mononuclear cells. Present drug treatments are largely ineffective against the organism when inside the cells.
The present invention is based on the surprising observation that leucocytes infected with HIV can be selectively removed from blood.
The present invention provides a method for the removal of infected, activated and/or defective leucocytes from blood, which comprises bringing blood that comprises infected, activated and/or defective leucocytes into contact with an adsorbent carrier that has a greater affinity for infected, activated and/or defective leucocytes than for uninfected, non-activated or non-defective leucocytes. If desired, the treated blood may be returned to the subject from whom it was obtained.
The method of the present invention effects selective adsorption of infected, defective and activated leucocytes including mononuclear cells i.e. monocytes/macrophages, and also activated lymphocytes, for example CD4+ lymphocytes, including resting CD4+ lymphocytes. Normal, that is to say, non-infected, non-activated and non-defective leucocytes do not appear to adhere to the adsorbent carrier. This is surprising, as the previous uses of apheresis were based on the fact that granulocytes or inflammatory cells, classified as granulocytes and monocytes, were selectively adsorbed in preference to lymphocytes. Contrary to the previous observations that selective adsorption is based on cell type, we have now found that infection of a leucocyte with a virus, which is an intracellular parasite, activation of a leucocyte and/or defects in a leucocyte brings about adsorption to an apheresis column regardless of the leucocyte type.
We presently believe that infection results in selective binding of a population of leucocytes to an adsorbent carrier. We believe that this is owing to a change in a property of the leucocytes, for example a change in the cell surface receptor population, for example a change in receptor types or receptor numbers. A change caused to a leucocyte by infection may be considered as activation of the leucocyte, or the leucocyte may be regarded as defective. However, leucocytes may be activated or rendered defective in other ways that result in the same or analogous selective binding as does infection. Infection, activation and/or defects may result in the same or similar cell surface changes, for example the changes may be in the same receptor type. However, this may not be the case, and the method of the invention is not dependant on the precise mechanism of the selective adsorption of the infected, activated and/or defective leucocytes.
Whilst the aforementioned is the current theory for the functioning of the invention, the applicant is not bound by this theory.
In a further embodiment of the invention immune recovery in a patient infected with a pathogenic organism is effected by contacting blood from the patient with a carrier having a contact angle to water within the range of from 55xc2x0 to 95xc2x0 and returning the blood to the patient. The absorbent carrier may be as described above, and is preferably cellulose acetate, especially in one of the preferred forms described above.
When the method of the invention is carried out, the above described effects relating to leucocyte removal and the immune recovery both occur.
Immune recovery denotes an improvement in any one or more aspects of the immune system. One aspect of immune recovery is an immunological improvement. HIV has a particularly profound effect on a subject""s immune system.
Another aspect of immune recovery is the immuno reconstitution that results from removal of leucocytes and their replacement by new leucocytes. Yet a further aspect of immune recovery may result from reduced generation of immunosuppressive cytokines (TNF-xcex1).
Certain aspects of immune recovery appear to result from removal of infected leucocytes whilst other aspects may be fully or partly independent of leucocyte removal.
In AIDS, the CD4 leucocyte count is regarded as indicative of the status of a subject""s immune system. When blood of an HIV positive person is contacted with a carrier as defined above the person""s CD4 count increases, thereby demonstrating an immune improvement, and hence immune recovery. The present invention also provides use of an adsorbent carrier described above in the manufacture of an agent or adsorbent for use in apheresis for the various aspects of immune recovery described above.
The invention further provides a method for the treatment of a patient having an infection that involves infected, activated and/or defective leucocytes, which comprises bringing blood from the patient into contact with a carrier that has a higher affinity for infected, activated and/or defective leucocytes than for uninfected non-activated or non-defective leucocytes and returning the treated blood to the patient.
Particularly, the leucocytes may be activated or infected leucocytes and the cells may be brought into contact with an adsorbent carrier that has a greater affinity for activated or infected leucocytes than for normal leucocytes.
More particularly, the leucocytes may be infected leucocytes and the cells may be brought into contact with an adsorbent carrier that has a greater affinity for infected leucocytes than for normal leucocytes.
Leucocytes may be infected with a virus or with another intracellular pathogenic organism, for example, an intracellular parasitic organism. Leucocytes may be infected with a virus that stimulates the cells and brings about an activation and/or a change in the surface receptors of the cells. For example, the virus is HIV or HCV, particularly HIV. HIV has a profound effect on cells of the immune system.
As explained above, HIV infects monocytes/macrophages and CD4+ lymphocytes. Different viruses may infect different populations of leucocytes. Monocytes/macrophages may be particularly subject to infection owing to their phagocytic activity. However, different viruses may infect different populations of lymphocytes. HIV infects CD4+ lymphocytes because the virus effects entry via the CD4 receptor. Other viruses may effect entry into lymphocytes via different receptors and hence infect the population of lymphocytes that possess that particular receptor.
The leucocytes may be infected with an intracellular parasitic organism which organism brings about an activation and/or a change in the surface receptors of the cells, for example, Mycobacterium leprae. Mycobacterium leprae infects monocytes in paricular.
The invention further provides the use of an adsorbent carrier that has a greater affinity for infected, activated and/or defective leucocytes than for normal leucocytes in the manufacture of an agent for the removal of infected, activated and/or defective leucocytes from blood.
The invention further provides a method for use in flushing out (purging) infected leucocytes from resting sites in a subject, which comprises bringing blood from the subject, which blood comprises infected leucocytes into contact with an adsorbent carrier that has a greater affinity for infected leucocytes than for normal leucocytes. The treated blood may be returned to the subject to flush out infected cells from resting sites. Such flushing out of cells is commonly known in the art as xe2x80x9cpurgingxe2x80x9d. The cells are especially leucocytes infected with a virus; for example, HCV or especially HIV. Such infected cells include mononuclear cells (monocytes/macrophages) and CD4+ lymphocytes. Flushing infected cells from resting sites results in the virus within the cells becoming vulnerable to drug therapy.
The invention further provides a method for use in the treatment of a disease in which infected, activated and/or defective leucocytes have a changed surface, which comprises bringing blood that comprises infected, activated and/or defective leucocytes into contact with an adsorbent carrier that has a greater affinity for infected, activated and/or defective leucocytes than for uninfected, non-activated or non-defective leucocytes. The treated blood may be returned to the patient to complete the treatment of the disease.
The types of diseases and disorders with which the present invention may be used include those in which infection of host cells, for example, with a virus or with another intracellular pathogenic, for example, parasitic organism, gives rise to an alteration of surface properties of the infected cells. For example, infection of cells of the immune system may give rise to activation of those cells, accompanied by a change in surface receptors.
The invention further provides a method for use in the treatment of a patient having a disease or disorder in which leucocytes are infected, which comprises bringing blood that comprises infected leucocytes into contact with an adsorbent carrier that has a greater affinity for infected, activated and/or defective leucocytes than for uninfected leucocytes. The treated blood may be returned to the patient to complete the treatement of the disease or disorder.
Still further, the invention provides a method for the treatment of a patient which method comprises bringing blood that comprises infected leucocytes from the patient into contact with an adsorbent carrier that has a greater affinity for infected, activated and/or defective leucocytes than for uninfected leucocytes and returning the blood to the patient.
Diseases which may be treated according to the present invention include leprosy, HCV infection and HIV infections, including ARC, AIDS and symptomless HIV infections. Still further, the invention provides the use of an adsorbent carrier in the preparation of materials for use in the above methods.
The invention further provides a method for effecting immune recovery in a patient infected with a pathogenic organism, particularly HIV, which comprises contacting blood from the patient with an adsorbent carrier that has a contact angle to water in the range between 55xc2x0 and 95xc2x0. For example, the patient may be infected with HIV.
In any embodiment of the present invention the adsorbent carrier may be contacted with plasma as an alternative to whole blood. Blood may be separated into plasma and other components in an appropriate manner before treatment according to the present invention. However, it is generally simpler and cheaper to treat whole blood. In general, therefore, plasma is used only in special circumstances.